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Fig. 5 Rabaptin-deficient dHL-60 cells fail to recycle endosomes, induce PAK phosphorylation, and F-actin polymerization at the leading edge. (A) Schematic illustrating how RABEP 1 facilitates endosomal signaling to activate PAK, promoting actin filament formation at the leading edge. (B - D) Representative images and quantification of the internalization rates of Transferrin (Tfn) and WGA. Scale bar, 10 μm. The average fluorescence intensity per cell was calculated and normalized to the 10-minute time point. The experiment included two biological repeats, each with 15- 31 cells per group at each time point. Data are shown as mean ± SD, analyzed with Dunnett's multiple comparisons test. (E- G) Representative images and quantification of recycling rates of Tfn and WGA. Scale bar, 10 μm. (C) The fluorescence intensity per cell was averaged and normalized to the 10-minute time point. (E)The fluorescence intensity per cell was averaged and normalized to the 0-minute time point. The assay was repeated twice with a total of 15-31 cells per group per timepoint. Data are presented as mean ± SD, analyzed with Dunnett's test. (H, I) Representative images and quantitative immunoblot analysis of PAK and pPAK in control and two separate RABEP 1 knockdown dHL-60 cells after fMLP stimulation at the indicated timepoints. VINCULIN served as a loading control, and the ratio of pPAK to PAK expression was quantified. The immunoblot included three biological repeats, with data shown as mean ± SD, analyzed by Dunnett's test. Significance levels are indicated by asterisks, where four asterisks denote p < 0.0001. (J, K) Representative images and quantification of polarization immunofluorescence staining in control, RABEP 1 knockdown with GFP, FL, or Δ5-2 RABAPTIN expressing dHL-60 cells. Scale bar, 10 μm. The experiment was performed twice, with 118- 226 cells in each group. Data are shown as mean ± SD, analyzed with Dunnett's test. (L, M) Representative images and quantification of actin fluorescence intensity in the same group of cells. Scale bar, 50 μm. Two biological replicates were conducted, with 31-35 cells per group. Data are presented as mean ± SD, analyzed with Dunnett's test.